Cocaine, amphetamine, methamphetamine, and methylenedioxy-methamphetamine (MDMA, or “ecstasy”) top the list of widely abused psychostimulants. The proposed mechanism of action of these drugs is through the inhibition (in the case of cocaine) or reversal (in the case of amphetamines) of transporter function of the plasma membrane monoamine transporters (MATs), including the human dopamine transporter (hDAT) and the human serotonin transporter (hSERT).
Like most members of the solute carrier 6A family (SLC6A), serotonin (SERT), norepinephrine (NET), and dopamine (DAT) transporters are expressed on the plasma membrane, where they catalyze the uptake of their respective substrates. They clear the neurotransmitters from the extracellular space following synaptic release, and serve as important regulators of the signal amplitude and duration at the monoaminergic synapses that ultimately drive behaviors. Recently, the structures of LeuT, a bacterial homologue of MATs, in a substrate-bound occluded, substrate-free outward-open, and an apo inward-open state and also with competitive and noncompetitive inhibitors have been determined. Together with computational modeling and experimental data gathered over the past decade, these structures have dramatically facilitated the understanding of several aspects of SERT, NET, and DAT transporter function, including the molecular determinants of ligand interaction. These data have also enabled identification of important structures for ligand interaction. However, the binding pockets of cocaine to hDAT and hSERT have not been clearly established. Some studies point to either overlapping pockets or neighboring pockets separated by a set of aromatic residues called the aromatic lid near the salt bridge formed between R85 (transmembrane domain 1; TMD1) and D476 (transmembrane domain 10; TMD10) in DAT.
SERT serves as an important regulator of the signal amplitude and duration at the serotonergic synapses that ultimately drive serotonin-associated behaviors. SERT is pharmacologically interesting, being a target of several clinically relevant drugs including the classical tricyclic antidepressants, newer selective serotonin reuptake inhibitors (SSRIs), and psychostimulants including cocaine, amphetamine and MDMA.
SERT cDNAs have recently been isolated from the human parasite Schistosoma mansoni, and are designated as SmSERT and SmDAT. In both transporters, transport activities of their endogenous substrates were identical to their mammalian counterparts. However, in SmSERT, serotonin, but not other substrates such as amphetamine and MDMA, induced efflux in the parasite carrier. The parasite SERT also displays dramatically reduced affinity for several inhibitors, including cocaine and SSRIs.
Preliminary efforts to understand the structure and function of SERT involved homology modeling of these transporters using the crystal structures of members of the major facilitator superfamily (such as lactose permease symporter) as template (Abramson et al., 2003, Science 301:610-615; Ravna et al., 2006, Bioorg Med Chem 14:666-675). This model explained the binding mode of several substrates including S-methamphetamine and SSRIs. Further modeling efforts involved the use of the leucine transporter (LeuT) from a thermophilic bacteria, Aquifex aeolicus (Yamashita et al., 2005, Nature 437:215-223), which has provided a suitable template to understand the ion binding sites (Forrest et al., 2007, Proc Natl Acad Sci USA 104:12761-12766) and orthosteric and allosteric binding sites in serotonin and other monoamine transporters (Kitayama et al., 1992, Proc Natl Acad Sci USA 89:7782-7785; Sulzer et al, 2005, Prog Neurobiol 75:406-433; Beuming et al., 2008, Nat Neurosci 11:780-789; Forrest et al., 2008, Proc Natl Acad Sci USA 105:10338-10343). Adding to the initially determined structure, structures of LeuT in a substrate-free outward-open; a substrate-bound occluded; and an apo inward-open state have been determined (Krishnamurthy and Gouaux, 2012, Nature 481:469-474). Structures of LeuT in complex with competitive and noncompetitive inhibitors are also available (Rudnick, 2006, J Membr Biol 213:101-110; Singh et al., 2007, Nature 448:952-956; Zhou et al., 2007, Science 317:1390-1393; Singh et al., 2008, Science 322:1655-1661; Zhou et al., 2009, Nat Struct Mol Biol 16:652-657). These LeuT structures were instrumental in developing homology models of SERT in both outward- and inward-open conformation and in determining the molecular interactions governing the ion binding, orthosteric, and psychostimulant binding sites (Rudnick, 2006, J Membr Biol 213:101-110; Celik et al., 2008, J Am Chem Soc 130:3853-3865; Manepalli et al., 2012, AAPS J 14:820-831). However, the binding pocket of psychostimulants to hDAT and hSERT has not been clearly established with some studies pointing to either overlapping pockets or neighboring pockets separated by a set of aromatic residues called the aromatic lid near the TM1-TM10 salt bridge formed between R85 and D476.
The hybrid structure-based (HSB) method (Kortagere and Welsh, 2006, J Comput Aided Mol Des 20:789-802) allows for screening small-molecule libraries and designing small-molecule inhibitors of therapeutically relevant targets. These include inhibitors of novel protein-protein interactions of the malarial parasite (Kortagere et al., 2010, J Chem Inf Model 50:840-849.), Toxoplasma gondii (Kortagere et al., 2011, J Comput Aided Mol Des 25:403-411; Kortagere, 2012, Expert Opin Drug Discov 7:1193-1206) and inhibitors that block the hexamerization of the HIV-1 capsid protein (Kortagere et al., 2012, Methods Mol Biol 929:359-375; Kortagere et al., 2012, J Virol 86:8472-8481). The HSB method was also instrumental in the design of atypical agonists of dopamine D3 receptor that bind to the receptor and modulate its function (Kuzhikandathil and Kortagere, 2012, Pharm Res 29:2264-2275). The key component of the HSB method is design of a three-dimensional pharmacophore using structural information derived from molecular dynamics (MD) simulations and known biochemical data, such as site-directed mutagenesis of key residues of the target protein. Hit molecules from the screening are subjected to several filtering schemes (Kortagere and Welsh, 2006, J Comput Aided Mol Des 20:789-802; Chekmarev et al., 2008, Chem Res Toxicol 21:1304-1314; Kortagere et al., 2008, Pharm Res 25:1836-1845; Kortagere et al., 2009; Kortagere et al., 2012, Methods Mol Biol 929:359-375; Kortagere et al., 2012, J Virol 86: 8472-8481) customized to every target. The best ranking compounds are then tested in in vitro assays.
Past studies have identified small molecule ligands that competitively inhibit the interactions of psychostimulants with receptors, transporters, ion channels or enzymes at their orthosteric site. However, these compounds have the potential to have psychostimulant properties. Thus, there is a need to design new compounds that are specific in their interaction with the transporters, efficacious in treating brain-related diseases or disorders, and free of side effects.
There is a need in the art for novel therapeutic agents that treat or prevent brain-related diseases or disorders involving aberrant monoamine signaling. The present invention fulfills this need.